Bacteria of the genus Chlamydia are obligate intracellular bacterial parasites. C. trachomatis is a human adapted species divided into several serologically different variants that cause serovar-specific diseases. While genital serovars of C. trachomatis cause approximately 100 million sexually transmitted infections worldwide annually, ocular serovars are a leading cause of preventable blindness in developing countries. All chlamydiae must transition between two terminally differentiated cell forms to complete their life cycle. Following host cell entry by the infectious, non-replicative Elementary Body (EB), the organism transitions to the replicative, non- infectious Reticulate Body (RB). Other pathogenic bacteria that transition between developmental forms can respond to nutrient availability as signaled via the stringent response (SR) as a regulator of differentiation. A typical SR is initiatd by synthesis of the RNA polymerase regulators guanosine tetraphosphate (ppGpp), also referred to as the alarmone, and the transcription factor DksA. Because deletion of dksA can disrupt differentiation of bacterial development, expression of dksA in C. trachomatis is consistent with a role for the SR in C. trachomatis development. With a genome of only 1.04 Mb, C. trachomatis lacks several predicted essential enzymes and entire metabolic pathways and must therefore obtain numerous nutrients, including amino acids and nucleotides, from the host cell. The proposed research is based on the hypothesis that C. trachomatis development is regulated by nutrient availability via the SR and that EB generation coincides with declining intracellular nutrient availability as the increasing bacterial burden depletes host nutrient pools Consistent with cell form-specific roles during infection and replication, comparison of EB and RB transcriptional activity during intracellular replication, analysis of cell form-specific proteomes, and biochemical analyses have revealed that EBs and RBs are metabolically specialized. As such, nutritional regulation of transitions between EB-RB and RB-EB may be triggered by different nutrients. Identification of such differential regulation will require direc comparison of both cell forms to the same nutritional cues. The goal for Specific Aim 1 is to determine if chlamydial SR components (e.g., dksA/DksA) are expressed coincident with pathogen morphological transitions and declining host nutrient pools during intracellular replication. In order to identify specific nutritional triggers for the C. trachomatis SR, the goalfor Specific Aim 2 is to measure molecular markers of the C. trachomatis EB and RB SR during starvation for specific nutrients. This project addresses the mission of the National Institutes of Health in that it will generate fundamental knowledge about a major human pathogen.